The proposed studies are aimed at elucidating details of how the milk protein Alpha lactalbumin regulates the synthesis of lactose in mammary tissue during lactation. The long range goals of the proposed research is to delineate how metal ions bound to the galactosyl transferase (GT) and Alpha lactalbumin (Alpha LA) moieties of lactose synthetase promote and modulate catalytic activity. We hope to be able to answer the questions: a) what metal ions occupy the binding sites on these two moieties of the complex under physiological condition? b) how do these metal ions influence complex formation? What role, if any, do they play in altering the Km's for monosaccharides? Equilibrium metal ion binding studies of GT and of Alpha LA will form the basis for similar studies of the complex. Measurement of the pH dependence of the association constant for binding of metal ions to Alpha LA will be used to identify liganding side chains in this protein. The effect of metal ion binding on complex formation will be determined using steady state and nanosecond decay polarization measurements. Chemical reactivity studies of Alpha LA and complexes of Alpha LA with enzyme will be used to determine: a) side chains in the protein which ligand metal ions. b) changes in conformation of the protein on binding of metal ions and on formation of the complex with enzyme. NMR and fluorescence energy transfer measurements will be used to establish spatial relationships between: a) metal binding sites and other structural moieties in the Alpha LA molecule. b) metal ion and substrate binding sites in GT and in GT moieties in the lactose synthetase complex.